In spite of detailed studies of the events involved in eicosanoid biosynthesis by either the 5-lipoxygenase or cyclooxygenase pathway, little is known concerning the phospholipid precursor which donates arachidonate for eicosanoid biosynthesis. This seemingly straightforward question is difficult to approach experimentally due to the complexities of phospholipid turnover and arachidonic acid remodeling in the intact cell. Furthermore, these are substantial analytical complexities in measuring one substance present in a complex mixture and the fact that biosynthesis of only small quantities of eicosanoids result after a physiologically relevant stimulus. A long term objective of the work in this project is to investigate the biochemical origin of prostaglandins, leukotrienes, and platelet activating factor in terms of the precursor phospholipids. A hypothesis has been suggested that a unique precursor exist within inflammatory cells and perhaps other cells in the lung. This unique precursor in 1-alkyl-2-arachidonyl-GPC liberates the free arachidonic acid and lyso-PAF following hydrolysis by phospholipase A2. The two products can themselves be processed into eicosanoids and PAF, respectively. Experiments are proposed to test the precursor role of alkyl-arachidonyl- GPC using a specific activity strategy from pulse labeling with either radiolabeled or stable isotope labeled arachidonic acid or correspondingly labeled lyso-PAF. Tandem mass spectrometry will be used to determine specific activity of arachidonate containing phospholipids and the time course of formation of eicosanoid products will be determined. Initial studies will be carried out with transformed mast cells using IgE-anti DNP and DNP-albumin as stimulus. The specific activities of the eicosanoid products will be compared to the specific activities of precursor arachidonyl containing phospholipids. Strategies to alter specific activities will be employed and are based upon time-dependent remodeling, use of specific inhibitors, and addition of labeled precursors. These studies will also be carried out in alveolar Type II epithelial cells and pulmonary endothelial cells as models of noninflammatory cells in the lung. Recent studies have suggested that cells depleted of arachidonate (and its precursor linoleic acid) synthesize little eicosanoids and PAF. Basic studies of ether lipid biosynthesis, phospholipid remodeling, and lipid mediator biosynthesis will be studied in cells depleted of arachidonic acid or supplemented with other polyunsaturated fatty acids such as Omega-3 fatty acids.